This project focuses on the molecular mechanisms by which an autonomous parvovirus enters and establishes infection in its host cell. Structural and genetic approaches will be used to explore dynamic, sequential interactions between virions of Minute Virus of Mice (MVM) and host cell factors during entry. These studies will contribute to our understanding of the host range constraints and pathogenesis of parvoviruses, the molecular mimicry they use to usurp host regulatory circuits, and their potential use as oncotropic vectors for tumor immunotherapy. Viewing the virion as an entry nanomachine, particular emphasis will be placed on dissecting the function of the N-terminus of the minor capsid polypeptide, VP1SR, which has been defined as a major effector domain in viral penetration of the cell. We will combine genetic dissection and NMR spectroscopy to explore the structure and folding properties of the VP1SR, and cryo-electron microscopy to delineate the pH- and energy-driven structural transformations of the viral capsid shell. In vivo tracking by FLAsH labelling, differential PCR and specific antibodies will be combined with site- directed mutagenesis and trafficking assays to assess the roles, in viral entry, of specific residues and peptide motifs identified within the VP1SR and in the 8A pore at the virion 5-fold vertex. The relationship between steps of entry and the tropism of particular viral strains will be investigated in both productive and restrictive virus:host interactions. The roles of virion and VP1SR signaling, WW-domain protein interaction, ubiquitylation and VPR targeting will be explored in endosomal escape, cytosolic trafficking and entry of the virion into the nucleus. The role of the NS2 non-structural proteins in both tissue specificity and oncoselectivity will be explored in cell culture and in vivo. Viral anti-tumor vectors, based on MVM, designed to elicit tumor-specific CD8+ cytotoxic effector cells, will be optimized for their ability to augment immunotherapy of neoplastic disease. Strategies to improve vector production will be pursued and combinatorial approaches used to select recombinant viruses with enhanced target cell specificity, which will be tested for improved efficacy as immunomodulatory vectors. The proposed research will explore parvoviral structure and entry, as they relate to the basis of viral allotropism and oncoselectivity. This understanding is a necessary prerequisite for the translational use of these viruses as vectors for the introduction of therapeutic immunomodulatory genes into human tumor cells for the treatment of neoplastic disease.